Biochemical characterization, molecular cloning and expression of laccases - a divergent gene family - in poplar

P. Ranocha, G.J. McDougall, S. Hawkins, R. Sterjiades, G. Bordieres, D. Stewart, M. Cabanes-Macheteau, A. Boudet, D. Goffner

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    Abstract

    The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H2O2-independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90-kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm.

    The cDNA clones corresponding to the 90-kDa and 110-kDa proteins, lac90 and lac110, were obtained by a PCR-based approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with gene-specific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.
    © John Wiley & Sons, Inc
    Original languageEnglish
    Pages (from-to)485-495
    Number of pages11
    JournalEuropean Journal of Biochemistry
    Volume259
    Issue number1-2
    DOIs
    Publication statusPublished - Jan 1999

    Keywords

    • laccase
    • poplar
    • lignification
    • dehydrogenation polymers

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    Ranocha, P., McDougall, G. J., Hawkins, S., Sterjiades, R., Bordieres, G., Stewart, D., Cabanes-Macheteau, M., Boudet, A., & Goffner, D. (1999). Biochemical characterization, molecular cloning and expression of laccases - a divergent gene family - in poplar. European Journal of Biochemistry, 259(1-2), 485-495. https://doi.org/10.1046/j.1432-1327.1999.00061.x